One of the fundamental questions in auditory and vestibular neurobiology examines how hair cells, the sensory receptors of the internal ear, deploy a transduction apparatus so sensitive that it detects deflections no larger than the width of a hydrogen atom. Experiments examining the mechanisms of mechanoelectrical transduction in hair cells have revealed that they employ Ca2+ to regulate adaptation, the process whereby hair cell reset their sensitivity to small displacements. Ca2+ sets the open probability of transduction channels at rest to 5-10% so that the channels can detect minute displacements around the hair bundle's, the organelle of mechanoelectrical transduction, ambient position. Ca2+ is also required for hair bundle twitches, which contribute toward high-sensitivity displacement detection. Despite the pivotal role of Ca2+ in mechanoelectrical transduction, hair bundles are exposed to extracellular fluid, endolymph, which has a bulk Ca2+ concentration o f 20-100mM , depending on the species. The exquisite sensitivity of hair cells is lost in vivo in 20-50mM Ca2+: isolated hair cells exhibit no adaptation and the resting open probability of transduction channels increased to more than 50%. Moreover, in an intact cochlea, it has been reported that the open probability of transduction channels at rest was about 5-10%. Simulation of the in vivo conditions, in vitro requires millimolar concentrations of Ca2+. This crucial role of Ca2+ in mechanoelectrical transduction and the apparent low bulk endolymphatic Ca2+ poses a paradox that has eluded auditory and vestibular physiologists for decades. Emerging preliminary biochemical, molecular, and physiological data suggest that hair cells may express the plasma membrane Ca2+ ATPase (PMCA), at high density in the hair bundle. We hypothesize that PMCA located at hair bundles, extrudes Ca2+ to maintain a relatively high Ca2+ concentration at the local domains of transduction channels to regulate adaptation and, support hair-cell sensitivity to small displacements. Ca2+ influx through voltage-gated Ca2+ channels at the basolateral membrane, and Ca2+ diffusion to the hair bundle, which is endowed with high density of PMCA, will setup a transcellular Ca2+ flux from the basolateral to the apical surface. The goal of this research project is to determine the role of PMCA in the establishment of low hair bundle Ca2+ and high local extracellular Ca2+ concentrations to ensure the atomic sensitivity of hair cells to mechanical displacement. To accomplish this goal, we are using specific antibodies to localize the Ca2+ pump in the isolated hair cells and by using quantitative immuniblot and immunoprecipitation analysis, we will determine the density of the protein at both the hair bundles and the basolateral membrane. We predict that the density of the protein in hair cells will be high and that under good electro-physiological recording conditions the current generated by the pump can be resolved and characterized using kinetic, and pharmacological criteria. By using the Ca2+ -sensitive probes, we are determining the Ca2+ fluxes at the apical and basolateral membrane and will ascertain the Ca2+ gradient established by the Ca2+ pump. In all these aims, we can determine the functional properties of the Ca2+ pump of isolated hair cells and semi-intact sacculi from the bullfrog, chick and mice.